U.S. patent application number 11/022510 was filed with the patent office on 2005-05-19 for agglutination assay method in porous medium layer.
Invention is credited to Kawasaki, Kazuya, Nagata, Masahito, Nakamura, Kentaro, Seshimoto, Osamu, Tanaka, Toru.
Application Number | 20050106757 11/022510 |
Document ID | / |
Family ID | 16374215 |
Filed Date | 2005-05-19 |
United States Patent
Application |
20050106757 |
Kind Code |
A1 |
Nakamura, Kentaro ; et
al. |
May 19, 2005 |
Agglutination assay method in porous medium layer
Abstract
An agglutination assay method for quantitatively determination
of an analyte in an aqueous liquid sample using particles bearing
an anti-analyte. The agglutination is conducted in the porous
medium layer of the analysis element. A speedy quantitative
analysis of the analyte can be conveniently attained with high
sensitivity. When the particle-labeled anti-analyte is contained in
the porous medium layer, the anti-analyte can be stored with higher
stability in the dry state. A dry analysis element for enabling
such analysis method is also provided.
Inventors: |
Nakamura, Kentaro; (Saitama,
JP) ; Kawasaki, Kazuya; (Saitama, JP) ;
Seshimoto, Osamu; (Saitama, JP) ; Nagata,
Masahito; (Tokyo, JP) ; Tanaka, Toru; (Tokyo,
JP) |
Correspondence
Address: |
REED SMITH, LLP
ATTN: PATENT RECORDS DEPARTMENT
599 LEXINGTON AVENUE, 29TH FLOOR
NEW YORK
NY
10022-7650
US
|
Family ID: |
16374215 |
Appl. No.: |
11/022510 |
Filed: |
December 22, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11022510 |
Dec 22, 2004 |
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10740243 |
Dec 18, 2003 |
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10740243 |
Dec 18, 2003 |
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10322847 |
Dec 18, 2002 |
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10322847 |
Dec 18, 2002 |
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10025133 |
Dec 19, 2001 |
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10025133 |
Dec 19, 2001 |
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09613478 |
Jul 11, 2000 |
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Current U.S.
Class: |
436/524 |
Current CPC
Class: |
G01N 33/525 20130101;
G01N 33/587 20130101; G01N 33/54346 20130101 |
Class at
Publication: |
436/524 |
International
Class: |
G01N 033/551 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 12, 1999 |
JP |
11-197419 |
Claims
What is claimed is:
1. An agglutination assay method for quantitative determination of
an analyte in an aqueous liquid sample using particles bearing an
anti-analyte, the anti-analyte being capable of specifically
binding to the analyte so as to cause agglutination of the
particles, comprising: supplying said sample, together with said
particles, to a porous medium layer to cause the agglutination of
said particles in said porous medium layer; and measuring the
extent of the agglutination of the particles in the porous medium
layer to determine the amount of the analyte in the sample.
2. The method according to claim 1, wherein said particle is a
colloidal metal and the extent of the agglutination of the
particles is detected from a change in color tone of the colloidal
metal caused by the agglutination.
3. The method according to claim 2, wherein said colloidal metal is
colloidal gold or colloidal silver.
4. The method according to claim 1, wherein said analyte is an
antigen and said anti-analyte is an antibody.
5. An agglutination assay method for quantitative determination of
an analyte in an aqueous liquid sample using particles bearing an
anti-analyte, the anti-analyte being capable of specifically
binding to the analyte so as to cause agglutination of the
particles, comprising: providing a porous medium layer containing
said particles; supplying said sample to the porous medium layer to
cause the agglutination of said particles in the porous medium
layer; and measuring the extent of the agglutination of the
particles in the porous medium layer to determine the amount of the
analyte in the sample.
6. The method according to claim 5, wherein said particle is a
colloidal metal and the extent of the agglutination of the
particles is detected from a change in color tone of the colloidal
metal caused by the agglutination.
7. The method according to claim 6, wherein said colloidal metal is
colloidal gold or colloidal silver.
8. The method according to claim 5, wherein said analyte is an
antigen and said anti-analyte is an antibody.
9. A dry analysis element for quantitative determination of an
analyte in an aqueous liquid sample by measuring the extent of
agglutination of particles bearing an anti-analyte, the
anti-analyte being capable of specifically binding to the analyte
so as to cause agglutination of the particles, comprising: a porous
medium layer containing said particles bearing the anti-analyte;
whereby, when the sample is applied to the porous medium layer, the
agglutination of said particles takes place in the porous medium
layer.
10. The dry analysis element according to claim 9, wherein the
porous medium layer has an optical characteristic so that a change
in color tone caused by the agglutination can be detected from
outside of the porous medium layer.
11. The dry analysis element according to claim 9, wherein the
porous medium layer is formed of a fibrous material.
12. The dry analysis element according to claim 11, wherein the
porous medium layer is formed of knitted cloth or woven cloth.
13. The dry analysis element according to claim 9, wherein the
porous medium layer is formed of a non-fibrous material.
14. The dry analysis element according to claim 9, wherein the
porous medium layer also serves as a spreading layer.
15. The dry analysis element according to claim 9, which further
comprises a support on which the porous medium layer is laminated
by means of partial adhesion.
16. The dry analysis element according to claim 9, wherein said
particle is a colloidal metal and the extent of the agglutination
of the particles is detected from a change in color tone of the
colloidal metals caused by the agglutination.
17. The dry analysis element according to claim 16, wherein said
colloidal metal is colloidal gold or colloidal silver.
18. The dry analysis element according to claim 9, wherein said
analyte is an antigen and said anti-analyte is an antibody.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to a method for detecting and
analyzing a trace substance by utilizing the agglutination assay,
in which an analyte reacts with a particle-labeled anti-analyte,
such as an antibody, to cause the particle agglutination.
Particularly, the present invention relates to a dry analysis
method for causing the agglutination of the particles bearing the
anti-analyte in a layer construction of a dry analysis element.
Also, the present invention relates to a dry analysis element which
enables such analysis method.
BACKGROUND OF THE INVENTION
[0002] In recent years, it has come to be very important to
quantitatively analyze a trace substance, particularly antibody or
antigen, in a specimen promptly, conveniently and precisely in
order to diagnose the condition of diseases or judge the effects of
treatment. For this purpose, widely employed has been an
immuno-serological test for assaying the existence of an antigen or
antibody in the body fluid, in which the antibody or antigen is
adsorbed and immobilized to insoluble carrier particles and the
resulting particles are reacted with the antigen or antibody.
[0003] The latex particles agglutination immunoassay is performed
routinely by mixing a suspension of antibody-coated latex particles
(sensitized latex) with a specimen on a glass plate. The latex
particles agglutinate, or fail to agglutinate, as a result of
interacting with the analyte antigen in the specimen. The extent of
the agglutination can be determined by visual inspection. This
assay makes it possible to semi-quantitatively analyze the antigen
in the specimen by diluting the specimen at various ratios similar
to another qualitative assay.
[0004] In Japanese Patent Publication Nos. 11575/1983
(corresponding to U.S. Pat. No. 4,118,192), 43138/1987
(corresponding to DE 2749956A1) and 55013/1987 (corresponding to DE
2749956A1), there is proposed a method, in which latex particles
having an antibody bound thereto is reacted with the antigen in the
sample and the amount of the agglutination of the latex particles
is determined optically by nephelometry. According to the proposed
method, an antigen or antibody has come to be analyzed
quantitatively by an automatic analyzer.
[0005] In addition, Unexamined Japanese Patent Publication (KOKAI)
Nos. 141665/1990, 94719/1994 and 213891/1994 disclose a method
wherein an antigenic substance is detected by measuring a change in
the absorbance upon the agglutination of the colloidal gold-labeled
antibodies.
[0006] The above-described immunoassays do not require B/F
separation and in this point, they are useful. The latex reagent
is, however, poor in storage stability, since it is in the liquid
form. In the colloidal gold agglutination, the colloidal gold
solution or dispersion is not suitable as a reagent because of poor
storage stability. A colloidal gold-labeled reagent in the
lyophilized form must be mixed with a dedicated solution upon
measurement, which makes the operation cumbersome. This method is
also accompanied with such a drawback as unsuitability for use in
the measurement of a small amount of a sample.
[0007] A so-called dry analysis method is, on the other hand,
superior in storage stability and convenient operation. The
so-called wet system (or solution system) comprises dissolving a
reagent to be used for the assay in an aqueous solvent, thereby
preparing the corresponding reagent solution, adding this reagent
solution to a sample to be analyzed and then measuring the color
reaction product by a calorimeter. On the other hand, the dry
analysis method comprises spotting an aqueous sample directly to a
dry analysis element, such as test piece, analytical slide or
analytical tape, having a reagent composition incorporated therein
in the dry form and effecting colorimetry of the color development
or color change occurring in the element. The dry system is
superior to the wet system using a reagent solution in convenient
operation and speedy assay.
[0008] A method for causing agglutination in the layer of a dry
analysis element, thereby directly detecting the existence of an
agglutinate itself in the layer construction has not yet been
proposed.
[0009] A dry analysis method, so-called solid phase
immuno-chromatography method has also been proposed (for example,
Unexamined Japanese Patent Publication (KOKAI) No. 5326/1997, which
corresponds to EP 0834741A1). This method utilizes a
chromatographic medium which is a liquid-permeable material serving
a capillary action. The liquid-permeable sheet such as filter paper
has a sample feeding zone and a detection zone, the sample feeding
zone containing an colloidal gold-labeled antibody, and the
detection zone containing an immobilized second antibody for
binding to the different epitope of the analyte antigen. The second
antibody is used as a capturing antibody. When a test sample
containing an analyte antigen is fed to the sample feeding zone
containing the colloidal gold-labeled antibody, the analyte antigen
reacts with the colloidal gold-labeled antibody to form an
immunocomplex. The formed complex diffuses and migrates to the
detection zone containing the immobilized second antibody, by the
capillary action of the chromatographic medium. The complex of the
antigen and colloidal gold-labeled antibody are captured by the
immobilized second antibody. The existence of the analyte antigen
is confirmed by detecting the color tone of the colloidal gold
which appears in the detection zone containing the capturing second
antibody. Since the reagent used in this method is maintained at
dry condition just before assay, it is excellent in storage
stability. However, it is a problem that the result is not
quantitative. Furthermore, in principle, the method is a sandwich
method in which a colloidal gold-labeled antibody is captured by a
second antibody through intervening analyte antigen, it is
necessary to use the permeable medium sheet having a sufficiently
large area so that an excessive colloidal gold-labeled antibody is
diffused and removed from the detection zone to which the capturing
second antibody is immobilized. This is the reason why the method
is called as immunochromatography method. Accordingly, a plenty of
liquid must be fed to the sheet, and it is necessary to use a large
medium sheet. In addition, the immunochromatography method requires
a long assay time, since it takes enough time for removal of an
excessive colloidal gold-labeled antibody from the capturing zone
by the capillary action.
[0010] Under such a circumstance, the present inventors have
attempted to search for a material capable of causing agglutination
in a layer medium of a dry analysis element. As a result, the
inventors have found that agglutination can be caused in a porous
medium layer, which is used as a spreading layer of an analysis
element, and measured quantitatively with good sensitivity. By
incorporating a labeling antibody into the porous medium layer,
storage stability of the reagent, which is an important
characteristic of a dry analysis element, is also successfully
attained.
SUMMARY OF THE INVENTION
[0011] The present invention has been accomplished in view of the
aforementioned circumstances, and a first object thereof is to
provide a dry analysis method for determining an analyte using an
agglutination of the particles bearing an anti-analyte, by which a
high sensitive analysis is ensured while using a simple operation
and reagent can be stored with excellent stability in the dry
state.
[0012] A second object of the present invention is to provide a dry
analysis element which can detect agglutination caused by the
reaction between an analyte and an anti-analyte labeled with a
labeling particle, thereby analyzing the analyte in a convenient
and highly sensitive manner.
[0013] The first object of the present invention is attained by an
agglutination assay method for quantitative determination of an
analyte in an aqueous liquid sample using particles bearing an
anti-analyte, the anti-analyte being capable of specifically
binding to the analyte so as to cause agglutination of the
particles, comprising:
[0014] supplying said sample, together with said particles, to a
porous medium layer to cause the agglutination of said particles in
said porous medium layer; and
[0015] measuring the extent of the agglutination of the particles
in the porous medium layer to determine the amount of the analyte
in the sample.
[0016] In the present invention, the agglutination of particles
bearing an anti-analyte (such as a colloidal metal-labeled
antibody, which is also referred to as labeling particle or
carrier) in voids of a porous medium layer. Since a liquid sample
can be kept in these voids of the porous medium layer, the
agglutination can be caused with a large amount of the liquid
sample per unit area. Therefore, the detection of the agglutination
with higher sensitivity is expected. Further, by incorporating
particles bearing an anti-analyte into the porous medium layer in
advance, the porous medium layer can be made dry state to an extent
not harmful to stability thereof upon storage. Upon analysis, the
porous medium layer is wetted with an aqueous test sample and
thereby provides a reaction field sufficient for causing
agglutination of labeling particles bearing an anti-analyte.
[0017] The labeling particles may be fed, together with an analyte
upon assay, to the porous medium layer. Alternatively, the
particles bearing an anti-analyte may be incorporated in the porous
medium layer in advance, and the particles bearing an anti-analyte
may be subjected to agglutination by the immunoreaction with an
analyte.
[0018] The agglutination caused in the porous layer is detected as
an optical change of the transmitted or reflected light. The
existence of the agglutinate and its amount may be detected as a
turbidity change in the porous layer medium, or as a change in
color tone of the labeling particle due to agglutination. The
porous layer preferably has an optical characteristics which allows
the detection of turbidity change, coloration, or change in color
tone from outside of the layer.
[0019] The second object of the present invention is attained by a
dry analysis element for quantitative determination of an analyte
in an aqueous liquid sample by measuring the extent of
agglutination of particles bearing an anti-analyte, the
anti-analyte being capable of specifically binding to the analyte
so as to cause agglutination of the particles, comprising:
[0020] a porous medium layer containing said particles bearing the
anti-analyte;
[0021] whereby, when the sample is applied to the porous medium
layer, the agglutination of said particles takes place in the
porous medium layer.
[0022] Preferably, the porous medium layer may serve as a so-called
spreading layer. The porous medium layer may be formed of a fibrous
or non-fibrous material which is used for a spreading layer.
BRIEF DESCRIPTION OF THE DRAWINGS
[0023] FIG. 1 is an illustration showing the layer structure of one
embodiment of the dry analysis element according to the present
invention; and
[0024] FIG. 2 is a graphic representation showing the results of
Example 1, more specifically, the calibration curve of dry analysis
elements of the slide 1 obtained in the Example 1.
DETAILED DESCRIPTION OF THE INVENTION
[0025] Analyte and Anti-Analyte
[0026] As an analyte or a substance to be analyzed in the present
invention, any substance can be used insofar as there exists a
specific binding partner or substance thereto in the nature or such
a substance can be prepared by chemical means. The anti-analyte,
i.e., specific binding partner or substance as used herein means a
substance which can specifically recognize and bind to the analyte
and at the same time, can be bound to a labeling carrier
particle.
[0027] Examples of the combination of an analyte and an
anti-analyte thereto include combinations of antigen and antibody,
a certain saccharide and lectin, biotin and avidin, protein A and
IgG, hormone and receptor thereof, enzyme and substrate, and
nucleic acid and complementary nucleic acid. In the
above-exemplified combinations, the analyte and the anti-analyte
may be reversed.
[0028] The most ordinary example is a combination of an antigen as
an analyte and an antibody as an anti-analyte. The antibody as an
anti-analyte may be either a polyclonal or monoclonal antibody.
Alternatively, a plurality of different antibodies can be used. No
particular limitation is imposed on the class of the antibody and
it does not matter whether it is IgG or IgM. It may be a fragment
of an antibody, for example, Fab, Fab' or F(ab').sub.2. When a
monoclonal antibody is employed as a specific binding substance, an
analyte antigen must have at least two same epitopes in order to
cause agglutination of a labeling particle having an antibody bound
thereon. Alternatively, at least two different antibodies which
bind to different epitopes of the analyte antigen, respectively,
may be bound to the labeling carrier particle. When the analyte
antigen is composed of plural sub-units, such as hemoglobin,
however, there is no need to use plural different monoclonal
antibodies. Binding plural molecules of single kind of monoclonal
antibody to the labeling carrier (particle), the agglutination of
the particles can be caused by the reaction with the analyte
antigen. At least two antibody molecules are preferably bound to
the labeling carrier (particle) for causing agglutination.
[0029] Labeling Particle
[0030] As a labeling carrier or particle for labeling by binding an
anti-analyte, any particle can be used insofar as it undergoes
agglutination as a result of reaction with the analyte and the
anti-analyte bound to the particle and the extent of the
agglutination falls within a detectable range. As the labeling
particle, those ordinarily employed for immuno-agglutination can be
used. Examples of the carrier particle include organic
high-molecular latex particles such as polystyrene or
styrene-butadiene copolymer, and metals such as colloidal metal.
The labeling particles (or colloid) are preferred to have an
average particle size falling within a range of 0.02 to 10 .mu.m.
When the particles have an excessively large particle size, optical
strength due to optical reflection or light scattering of the
particle itself prior to the immunoreaction becomes too high,
resulting in difficulty in measurement of the change of the optical
density. Too small particle sizes, on the other hand, tend to lower
the detection sensitivity of the agglutinate.
[0031] Any conventionally known colloidal metal can be used as a
labeling particle. Examples include colloidal gold, colloidal
silver, colloidal platinum, colloidal iron and colloidal aluminum
hydroxide. In particular, colloidal gold and colloidal silver are
preferred because they color red and yellow, respectively, at a
proper particle size. The particle size of a colloidal metal is
preferably about 1 to 500 nm. The size of 5 to 100 nm is
particularly preferred, because it permits development of a strong
color tone.
[0032] The colloidal metal and the anti-analyte can be bound in a
conventionally known manner (for example, The Journal of
Histochemistry and Cytochemistry, Vol. 30, No. 7, pp 691-696
(1982)). For example, a colloidal metal and an anti-analyte (e.g.,
an antibody) are mixed in a proper buffer solution and incubated at
room temperature for at least 5 minutes. The reaction mixture is
centrifuged to remove a supernatant. The obtained precipitate is
dispersed into a solution containing a dispersant such as
polyethylene glycol to obtain an aimed colloidal metal bearing an
anti-analyte.
[0033] In the case of using a colloidal gold particle as the
colloidal metal, commercially available one may be employed.
Alternatively, a colloidal gold particle can be prepared according
to a conventional method, for example, a method of reducing
chloroauric acid with sodium citrate (Nature Phys. Sci., Vol. 241,
20(1973) etc.).
[0034] Layer Structure of Dry Analysis Element
[0035] FIG. 1 shows the layer structure of one embodiment of the
dry analysis element according to the invention. In FIG. 1,
reference numeral 10 designates a support on which a porous medium
layer 12 is laminated. The porous medium layer is also referred to
as porous layer, hereinafter.
[0036] The support 10 may be light non-transmitting (opaque),
light-semi-transmitting (translucent), or light-transmitting
(transparent), and it is generally preferable that the support is
light-transmitting and water-impermeable. Preferable materials for
the light-transmitting and water-impermeable support are
polyethylene terephthalate and polystyrene. In general, an
undercoating is provided or the support is subjected to
hydrophilization treatment in order to firmly adhere the porous
medium layer 12 to be laminated thereon.
[0037] The porous medium layer 12 may be fibrous or non-fibrous. As
the fibrous material, filter paper, non-woven cloth, woven cloth
(e.g., plain woven cloth such as broad and poplin), knitted cloth
(e.g., knitted cloth such as tricot, double tricot, and milaneaze)
or filter paper made of glass fibers may be used. Examples of the
non-fibrous material may be either one of a membrane filter
composed of cellulose acetate as described in Unexamined Japanese
Patent Publication (KOKAI) No. 53888/1974 (corresponding to U.S.
Pat. No. 3,992,258), or a particulate structure layer containing
interconnected voids and composed of inorganic or organic fine
particles as disclosed in Unexamined Japanese Patent Publication
(KOKAI) Nos. 53888/1974 (corresponding to U.S. Pat. No. 3,992,258),
90859/1980 (corresponding to U.S. Pat. No. 4,258,001) and
70163/1983 (corresponding to U.S. Pat. No. 4,486,537). A laminated
structure made of partially bonded multiple porous layers may also
be preferably used, examples of such structure being disclosed in
Unexamined Japanese Patent Publication (KOKAI) Nos. 4959/1986
(corresponding to EP 0166365A), 116258/1987, 138756/1987
(corresponding to EP 0226465A), 138757/1987 (corresponding to EP
0226465A) and 138758/1987 (corresponding to EP 0226465A).
[0038] The porous medium layer 12 may be a spreading layer having a
so-called metering function to spread, in the layer, a liquid over
an area substantially in proportion to the volume of the liquid fed
thereto. By providing the porous layer 12 with the function of a
spreading layer, the volume of the liquid fed to and spread in the
porous layer per area is made uniform and thereby accuracy at
quantitative determination of the analyte by agglutination in the
porous layer is improved.
[0039] Preferable materials for the spreading layer are woven and
knitted fabrics. The woven fabrics or like may be subjected to the
glow discharge treatment as described in unexamined Japanese Patent
Publication (KOKAI) No. 66359/1982 (corresponding to U.S. Pat. No.
4,783,315 and GB 2,087,974A). In order to adjust the area or rate
for spreading, the spreading layer may contain a hydrophilic
polymer or a surfactant as described in Unexamined Japanese Patent
Publication (KOKAI) Nos. 222770/1985 (corresponding to EP
0162301A), 219397/1988 (corresponding to DE 3717913A), 112999/1988
(corresponding to DE 3717913A) and 182652/1987 (corresponding to DE
3717913A).
[0040] In the porous medium layer 12, a buffer may be incorporated
so that the specific binding reaction between the particle-labeled
anti-analyte and the analyte occurs at an optimum pH. When the
binding reaction is an antigen-antibody reaction, for example, pH
buffers usable for an ordinary antigen-antibody reaction can be
employed. Specific examples of such buffers include buffer reagents
containing tris(hydroxymethyl)aminomethan- e (Tris), buffer
reagents containing phosphate, buffer reagents containing borate,
buffer reagents containing citric acid or citrate, buffer reagents
containing glycine, buffer reagents containing Bicine, buffer
reagents containing HEPES, and buffer reagents containing Good's
buffer agent such as MES (2-morpholinoethanesulfonic acid). The
reaction may be effected at any pH insofar as the pH is within a
range permitting an ordinary antigen-antibody reaction.
[0041] Further, in the porous layer 12, a high molecular polymer
such as polyvinyl alcohol, polyvinyl pyrrolidone or PEG
(polyethylene glycol) may be incorporated for the purpose of
promoting agglutination.
[0042] The porous layer 12 may contain particles bearing an
anti-analyte in advance. In this case, agglutination can be caused
in the porous layer 12 by simply applying a liquid sample
containing an analyte to the porous layer 12.
[0043] In addition, the porous layer 12 may contain light
reflecting fine particles of, for example, titanium dioxide or
barium sulfate so as to serve a light reflecting function. The
porous layer 12 having the light reflecting or light shielding
function may act as a white background so that change of color or
color density caused by agglutination in the porous layer 12 is
reflectively measured from the light-transmitting support 10 side.
When the porous layer 12 itself possesses an optical property
suitable for white background, the porous layer may not contain
light-reflecting fine particles.
[0044] The porous layer 12 may be laminated on the support 10 via
an undercoating layer (such as gelatin) formed on the support 10.
At this point, partial adhesion or spot-bonding may be applied to
the lamination. The terminology "partial adhesion" as used herein
is a definition of the adhesion feature or mode between adjacent
porous layers or between a porous layer and an adjacent non-porous
layer, and means "an adhesion at the interface between adjacent two
layers which are bonded substantially intimately to form an
integral structure by adhesion spots arranged partially or
discontinuously on the interface so as not to hinder substantially
uniform passage of a liquid through and between the adjacent two
layers" as in Unexamined Japanese Patent Publication (KOKAI) Nos.
4959/1986 (corresponding to EP 0166365A) and 138756/1987
(corresponding to EP 0226465A).
[0045] In general, an adhesive is spotted partially on the support,
and the porous layer is placed thereon to be adhered by uniformly
pressing with a light pressure. An adhesive may be spotted or
partially applied on the support by any of processes as disclosed
in Unexamined Japanese Patent Publication (KOKAI) Nos. 4959/1986
(corresponding to EP 0166365A), 138756/1987 (corresponding to EP
0226465A) and 23160/1989 (corresponding to DE 3721236A). A printing
process is preferred than other processes. An example of the
printing process is described in "INSATSU KOGAKU BINRAN" ("Handbook
of Printing Technology"), edited by Japan Printing Institution and
published by GIHO-DO SYUPPAN K.K. in 1983, pp 838-858. A silk
screen may be employed as in the following Examples.
[0046] Known adhesives disclosed in Unexamined Japanese Patent
Publication (KOKAI) No. 138756/1987 (corresponding to EP 0226465A)
and on pages 839 to 853 of the aforementioned "INSATSU KOGAKU
BINRAN" may be used. Examples of adhesive which may be used include
water-soluble adhesives, adhesives soluble in organic solvents and
thermally thermoplastic or thermosetting adhesives (hot-melt type
or heat sensitive adhesives). Specific examples of the
water-soluble adhesive are aqueous adhesives such as starch;
aqueous solutions of dextrin, carboxymethyl cellulose and polyvinyl
alcohols; and emulsion of copolymer of vinyl acetate and butyl
acrylate.
[0047] In the analysis element of the present invention, partially
bonding the porous layer 12 and the support 10 together does not
ensure the uniform passage of a liquid between the layers. However,
between the support and the porous layer, there exist
interconnected minute spaces which are not filled with an adhesive.
It is assumed that such spaces exhibit a sort of weak water
absorbing effect. When an aqueous liquid sample is spotted on the
porous layer, a liquid capable of permeating through and diffusing
into the porous layer can sufficiently reach the interface between
the support and the porous layer. Along with the diffusion and
permeation of the liquid, an agglutinate of labeling particles
formed in the porous layer can migrate to the interface
efficiently. Therefore, by choosing the interface between the
support and the porous layer as a measuring object, an optical
change caused by agglutination can be measured with good
sensitivity.
[0048] Preparation of Dry Analysis Element
[0049] The dry analysis element of the invention may be prepared by
any of the known processes described in the specifications of the
aforequoted patents. The analysis element of the invention may be
cut into a square piece having sides each ranging from about 15 to
30 mm or a disk having a substantially same area. It is preferred,
in view of the preparation, packaging, shipping, storage and
measuring operations, that the element be contained in a slide
frame as descried, for example, in Japanese Patent Publication No.
28331/1982 (corresponding to U.S. Pat. No. 4,169,751), Unexamined
Japanese Utility Model Publication No. 142454/1981 (corresponding
to U.S. Pat. No. 4,387,990), Unexamined Japanese Patent Publication
No. 63452/1982, Unexamined Japanese Utility Model publication No.
32350/1983 and Unexamined Japanese Patent publication No.
501144/1983 (corresponding to International Publication WO
83/00391) for use as a slide for chemical analysis. For the
convenience in some uses, it may be formed in a long tape shape
which is contained in a cassette or magazine, or a small piece
thereof may be applied on or contained in a card having an
opening.
[0050] Analysis Method Using the Dry Analysis Element
[0051] The analysis element of the invention may be used for the
quantitative analysis of an analyte in a sample liquid by using it
through the operations described in the specifications of the
aforequoted patents. When the analyte is an antigen or an antibody,
about 5 .mu.L to about 30 .mu.L, preferably 8 .mu.L to 15 .mu.L, of
an aqueous sample liquid such as plasma, serum or urine is spotted
on the porous medium layer 12. The analysis element thus spotted is
then incubated at a constant temperature of from about 20.degree.
C. to about 45.degree. C., preferably at a constant temperature of
from about 30.degree. C. to about 40.degree. C., for 1 to 10
minutes. The reflection optical density of the color or the change
in color in the element may be measured from the light-transmitting
support side, and the quantity of the analyte contained in the
sample can be determined using a preliminarily prepared calibration
curve based on the principle of colorimetry. The volume of the
spotted liquid sample and the time and temperature for incubation
are maintained constant to improve the accuracy in quantitative
analysis.
[0052] The measuring operation may be carried out while using the
chemical analysis apparatuses described in Unexamined Japanese
Patent Publication Nos. 125543/1985, 220862/1985, 294367/1986 and
161867/1983 (corresponding to U.S. Pat. No. 4,424,191) to realize
quantitative analysis at a high accuracy by extremely easy
operation. Depending on the purpose or required precision, however,
semi-quantitative measurement may be conducted by visually judging
the degree of coloring or change of color tone.
[0053] When the analysis element does not contain the labeling
particles bearing an anti-analyte, a necessary immunological
reaction can be carried out in a proper reaction mixture other than
the element, and then the resultant reaction mixture is spotted on
the element. Thus the analyte can be analyzed. For assaying an
antigen, for example, an aqueous sample liquid is mixed with a
solution containing an antibody labeled with the labeling particle
to complete the binding reaction, and then spotted on the
element.
[0054] For example, when an antigen, an antibody and a colloidal
metal are used as an analyte, an anti-analyte and a labeling
particle, respectively, a dry analysis element can be prepared and
a dry analysis using the element can be carried out as described
below.
[0055] Specifically, an antibody labeled with a colloidal metal is
dispersed in a buffer solution containing a proper dispersant or
stabilizer. The resulting dispersion is applied to a porous medium
layer, which is a spreading layer medium, and dried. The porous
layer is then laminated on a light-transmitting support 10 to
prepare a dry analysis element for agglutination. Alternatively,
after the lamination of the porous spreading layer on the
light-transmitting support, a solution containing an antibody
labeled with a colloidal metal is applied to the spreading layer
and dried to prepare a dry analysis element for agglutination.
[0056] An aqueous liquid sample containing an analyte (e.g.,
antigen) is spotted and applied onto the resulting analysis
element. The analyte causes the antigen-antibody binding reaction
with the colloidal metal-labeled antibody in the porous layer 12,
resulting in agglutination of the colloidal metal.
[0057] Agglutination changes the color tone or hue of the colloidal
metal so that the analyte in the sample can be detected and
quantitatively analyzed by measuring a change in the color tone of
the porous layer 12. For example, a colloidal gold before
agglutination colors reddish violet having a main absorption
wavelength at about 540 nm. By the agglutination, the colloidal
gold increases in size, leading to shifting of its absorbance to
the side of a longer wavelength, and as a result, the agglutinated
colloidal gold colors pale reddish purple or gray. Accordingly, the
analyte (antigen) can be quantitatively analyzed from a decrease in
the reflection optical density at 540 nm, an increase in the
reflection optical density at about 650 nm which appears by
agglutination, or a difference between reflection optical densities
at 540 nm and 630 nm.
EXAMPLE 1
Preparation and Evaluation of Dry Analysis Element
[0058] On a colorless transparent smooth and flat polyethylene
terephthalate (PET) film (support, thickness: 180 .mu.m)
undercoated with gelatin was placed a silk screen (pore size: 200
.mu.m, space between centers of pores: 700 .mu.m), to which an
adhesive for office job (starch paste) was then applied by means of
the squeeze method, followed by peeling off the screen to form mesh
points of the adhesive on the support. Then, thereon was placed a
white broad woven cloth made of a polyester which had been
previously immersed in 10 mM phosphate buffer (pH 7.2; supplemented
with 1.0% bovine serum albumin) at room temperature for 24 hours
and dried. The cloth was pressed and adhered by applying slight
pressure to form a spreading layer.
[0059] An aqueous solution of the following composition was coated
on the spreading layer and dried to form a reagent layer. The
respective components had the coverage as set forth below.
1 50 mM sodium phosphate buffer (pH 7.0) 242.3 g/m.sup.2 colloidal
gold-labeled 200 mg/m.sup.2 anti-human hemoglobin antibody
[0060] As the colloidal gold-labeled anti-human hemoglobin
antibody, there was used a colloidal gold antibody reagent
(conjugate of colloidal gold and mouse monoclonal anti-human
hemoglobin antibody) of an immunological kit for detecting fecal
occult hemoglobin "Immuno-Gold Hem" (produced by Godo Shusei Co.,
Ltd., sold by Wako Pure Chemicals Industries Ltd.).
[0061] Then, the thus prepared analysis element was cut into
rectangular chips of 12.times.13 mm size. The chips were encased
with slide frames described in Unexamined Japanese Patent
Publication No. 63452/1982 to prepare a dry slide 1 for analysis of
hemoglobin according to the present example.
[0062] A human hemoglobin A.sub.0 (Hb) (product of Exocell. INC)
was diluted with 0.2 M ammonium chloride aqueous solution (pH 6.8)
containing 6% polyethylene glycol 6000 to prepare a series of
diluted solutions of 0, 100, 250, 500 and 1000 ng/mL. The series of
diluted solutions was spotted onto the dry slide 1 in an amount of
20 .mu.L each. After each slide was incubated at 37.degree. C. for
5 minutes, the reflection optical density at central wavelength of
540 nm was measured from PET support side. FIG. 2 shows the
calibration curve obtained.
[0063] As apparent from FIG. 2, the slide 1 of the present example
exhibited that hemoglobin could be quantitatively determined at a
high accuracy. Especially, the decrease of OD.sub.540 was drastic
at a low Hb concentration range. This fact shows that the slide 1
of the present example is suitable for quantitative determination
of the analyte in lower concentration range and allows more highly
sensitive analysis. In addition, it is confirmed that the slide 1
of the present example allows a liquid sample to be kept in the
spreading layer (porous layer) upon spotting the liquid sample and
thus operability is superior owing to no disturbance of a liquid
flow at handling, at transportation to a measuring equipment, or at
slide transportation within a measuring equipment.
EXAMPLE 2
Storage Stability of Dry Analysis Element
[0064] The storage stability of the dry analysis element (slide 1)
obtained in Example 1 was examined. A dry analysis element is
generally stable at 4.degree. C. for a duration of about 1 year. In
this experiment, the elements were stored in a dry incubator set up
at 35.degree. C. for 0, 1, 4, 7 days after preparation of the
slides as an acceleration test.
[0065] As a comparative example, 250 .mu.g/mL of colloidal
gold-labeled anti-human hemoglobin antibody solution (50 mM sodium
phosphate, pH 7.0) was prepared and used as a reagent for
solution-type agglutination in the comparative example. The
solution reagent of the comparative example was stored in an
incubator of 35.degree. C. for 0, 1, 4, 7 days after the
preparation in a similar manner of the slide 1.
[0066] 100 ng/mL or 500 ng/mL of a human hemoglobin solution (human
hemoglobin A.sub.0 (Hb) (product of Exocell. INC), 6% polyethylene
glycol 6000, 0.2 M ammonium chloride (pH 6.8)) was spotted, in an
amount of 20 .mu.L, onto each slide 1 after storing for the
prescribed days. After each slide was incubated at 37.degree. C.
for 5 minutes, the reflection optical density at 540 nm was
measured from the support side. From the reflection optical density
obtained, hemoglobin concentration was calculated based on the
calibration curve made in Example 1 at the day when the slide 1 had
been prepared.
[0067] As for the comparative example, 100 .mu.L of the solution
reagent stored in the prescribed days was mixed with 50 .mu.L of
100 or 500 ng/mL of the human hemoglobin solution. After the
reaction for 10 minutes, the transmission optical density at 540 nm
was measured and hemoglobin concentration was calculated based on
the calibration curve made by using standard solutions in
advance.
[0068] As shown in Table 1, in the case of the solution reagent
(the comparative example) to be employed for conventional
solution-type colloidal gold agglutination, error of the calculated
Hb concentration became larger during storage at 35.degree. C. with
passage of days. The error reached to about 20% at the fourth day
and about 40% to 70% at the seventh day from the beginning of the
storage. On the other hand, the error was about 5% to 10% at the
fourth day and only 5% to 16% even at the seventh day from the
beginning of the storage in the case of the slide 1. As shown in
the above, the dry analysis element according to the present
invention is excellent in storage stability.
2TABLE 1 Comparison of Storage Stability Calculated Hb conc.
(ng/mL) Hb conc. Storage time Comparative (ng/mL) (day) Slide 1
Example 100 0 100 100 1 95 105 4 105 120 7 105 140 500 0 500 500 1
480 550 4 550 600 7 580 850
[0069] As described above, by the analysis method of the present
invention, the agglutination of an analyte (e.g., antigen) with
particles bearing an anti-analyte (e.g., colloidal gold-labeled
antibody) can be caused in the porous medium layer of the dry
analysis element. Since the agglutination is caused in the porous
medium layer capable of keeping a large amount of a liquid sample
per unit area, a speedy quantitative analysis of the analyte by the
particle agglutination can be conveniently attained with high
sensitivity.
[0070] In addition, the dry analysis element of the present
invention can be a medium of dry state, upon storage, to an extent
not harmful to stability of the reagent composition to be used.
Upon analysis, the porous medium layer is wetted by an aqueous
liquid sample and thereby can provide a reaction field to
sufficiently cause agglutination of particles bearing an
anti-analyte.
[0071] Furthermore, when particles bearing an anti-analyte are
incorporated in the porous layer in advance, a highly sensitive dry
analysis of the analyte can be conducted by simply spotting and
feeding a liquid sample containing the analyte. As compared with
the wet process agglutination using a conventional reagent
solution, an extremely speedy and convenient analysis can be
realized since preparation of the reagent at use and mixing
operation are not necessary. In addition, since a liquid sample is
received in the porous layer, excellent operability is ensured
owing to no disturbance of a liquid flow at transportation in
analysis operation.
* * * * *